Microalga-virus-virophage coculture reveals co-infection of multi-virophages with a giant virus

Virophages parasitize the replication of co-infecting giant viruses within eukaryotic cells, forming tripartite cell-virus-virophage (CVv) systems. Tripartite interactions are well-documented in protozoa, yet comparable systems in algae remain largely unexplored at the experimental level. Here, we report an experimentally validated CVv system involving the green, single-celled microalga Chlorella sp. DSL01, Dishui Lake large algal virus 1 (DSLLAV1), and multiple Dishui Lake virophages (DSLVs). Inoculation of Chlorella sp. DSL01 at low MOI established laboratory co-cultures in which time-series PCR detected DSLLAV1 early but not after Day 10, whereas all tested virophages persisted. Metagenomic profiling of the terminal supernatant (end-point sample) indicated a virophage-dominated assemblage with DSLV3 most represented. Droplet digital PCR at discrete time points (Days 5, 10, and 15) then provided absolute counts for DSLLAV1 and DSLV1/3/7, corroborating an early DSLLAV1 peak followed by collapse and/or a delayed rise of multiple virophages coincident with host growth recovery. Nested PCR on the algal pellet detected virophages DSLV1/3/7 but not DSLLAV1. Together, these results demonstrate that Chlorella sp. DSL01 supports co-infection by DSLLAV1 and multiple virophages, establishing an experimentally validated algal CVv system and revealing multi-virophage participation in freshwater algal virus-virophage-host dynamics.